JP2005315747A - New assay method of substance for controlling migration outside of rev protein kernel - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new assay method of a substance for controlling migration outside of an Rev protein kernel. <P>SOLUTION: A DNA originating from HIV is transfected to a cell, and the quantity of a structural protein p24 that manifests itself as a result is determined by an ELISA method, thus evaluating the inhibition activity of migration outside of a Rev kernel in a substance to be inspected. In this case, a cell sample having an inhibition rate of 0% and that having an inhibition rate of 100% are used contrastively, and both of them are compared, thus quantitatively evaluating the inhibition activity of migration outside of the Rev kernel of the substance to be inspected. And luciferases genes are transfected to each cell simultaneously with a DNA originated from HIV, a rough protein liquid extracted from each cell with the luciferases genes as a reference is diluted, and the amount of generation of p24 is measured in each weak diluted solution. A result for showing an improved quantity determination properties and reproducibility was obtained for the inhibition activity of transition outside of the Rev kernel of the substrate to be inspected by the assay method. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a novel assay method for a Rev protein nuclear translocation regulator and its use, and is preferably used to search for an anti-HIV drug whose action mechanism is inhibition of Rev protein nuclear translocation. it can.

  The number of AIDS patients, that is, those infected with HIV (Human Immunodeficiency Virus), has been increasing worldwide since the discovery, and the establishment of an immediate treatment is essential. As chemotherapy at the present stage, a method using a reverse transcriptase inhibitor and a protease inhibitor in combination is generally employed.

  A reverse transcriptase is an enzyme possessed by HIV that is necessary for translating viral RNA into DNA, and examples of inhibitors include AZT.

  Protease is an enzyme required after viral protein translation, and saquinavir or the like is used as its inhibitor.

  By the way, HIV gene codes Rev protein which is RNA binding protein. This Rev protein has a nuclear export signal (NES), is necessary for the export of viral mRNA, and can thus be said to be an essential protein for HIV replication / proliferation. Leptomycin B (LMB), valtrate, and the like have been reported as substances that inhibit / suppress the function of the Rev protein (see Non-Patent Documents 1 and 2 below).

Wolff, B. et al., Chemistry & Biology, 1997, 4, pp. 139-147 Murakami, N. et al., Bioorg. Med. Chem. Lett., 2002, 12, 2807-2810

  As described above, the Rev protein is an HIV-derived protein, and after binding to the HIV gene in the nucleus, it moves to the outside of the nucleus, and the HIV gene is translated to produce a virus constituent protein. Since the Rev translocation of the Rev protein is essential for the proliferation of HIV, the Rev protein has attracted attention as a target for HIV suppression.

  Conventionally, detection of Rev translocation of Rev protein has been performed using an anti-Rev antibody (the experimental method described in Non-Patent Document 1 also employs an experimental system using an anti-Rev antibody). However, since the commercially available anti-Rev antibody is not sensitive and has a large lot difference, and when the anti-Rev antibody is prepared from an animal, there is a variation in purity and sensitivity, so that the experimental results are reproducible. There was a difficult drawback. In addition, there remains a problem in terms of quantitativeness.

  Since the nuclear translocation of the Rev protein is essential for the growth of HIV, the detection of a substance that controls the nuclear translocation using the detection method can be accurately detected by detecting the movement of the Rev protein out of the nucleus with high accuracy. May be a stepping stone. Therefore, it is necessary to construct an assay method for a Rev protein nuclear translocation regulator with good accuracy.

  The present invention has been made in view of the above problems, and an object of the present invention is to provide a novel assay method for a Rev protein nuclear translocation regulator.

  As a result of diligent research in view of the above problems, the present inventor constructed a novel Rev protein nuclear translocation regulator assay method using a simple method such as ELISA without using an anti-Rev antibody, As for the quantitative property, good reproducibility was confirmed, and the present invention was completed.

That is, the present invention includes the following inventions A) to L) that are industrially and medically and medically useful.
A) After transfection with HIV-derived DNA, the expression level of a predetermined HIV-constituting protein is measured in the cells to which the test substance is administered, and the Rev export activity of the test substance is quantified based on the measurement result. A method for assaying a Rev nuclear export control substance, characterized in that
B) The assay method according to A) above, wherein the expression level of p24 is measured as a predetermined HIV constituent protein.
C) The assay method according to B) above, wherein the expression level of p24 is measured by an ELISA method.
D) The RRE region is transcribed into RNA in the host cell, and the Rev protein is not expressed in the first group of cells transfected with HIV-derived DNA that expresses the Rev protein, or the original function is lost. The second group of cells transfected with the HIV-derived DNA expressing Rev protein, and the third group of cells to which the test substance was administered after the same HIV-derived DNA as the first group of cells was transfected. The expression level of a predetermined HIV constituent protein is measured for each of the cells, and the Rev nuclear export inhibitory activity of the test substance is quantitatively evaluated based on the measurement results. Migration control substance assay.
E) The assay method according to D) above, wherein the expression level of p24 is measured as a predetermined HIV constituent protein.
F) The assay method according to E) above, wherein the expression level of p24 is measured by an ELISA method.
G) Characteristically evaluating the Rev. activity of the test substance in the nuclear export by comparing the two groups with the inhibition rate of 0% for the cells of the first group and 100% of the cells of the second group. The assay method according to D) above.
H) For each group of cells, a foreign gene is introduced in addition to the HIV-derived DNA, and a protein extract from each group of cells is diluted according to the expression level of the foreign gene. The assay method according to the above G), wherein the expression level of an HIV constituent protein is measured.
I) The assay method according to H) above, wherein a luciferase gene is introduced as a foreign gene.
J) The assay method according to H) or I) above, wherein an HIV-derived DNA and a foreign gene are introduced into a cell using a plasmid, and a plasmid for adjusting the transfection amount is further introduced into each group of cells.
K) An assay kit used in the assay method according to any one of A) to J) above.
L) An assay device used in the assay method according to any one of A) to J) above.

  The present invention provides a novel assay method for a Rev protein nuclear translocation regulator that exhibits good quantification and reproducibility that cannot be achieved by conventional methods. By utilizing the assay method of the present invention, HIV It becomes possible to efficiently discover a novel substance that controls the nuclear export of Rev protein, which is the key to suppression.

  Hereinafter, specific embodiments of the present invention will be described in more detail.

[1] Assay method for Rev protein nuclear translocation regulator of the present invention As described above, the present invention relates to the expression level of a predetermined HIV constituent protein in a cell administered with a test substance after transfection with HIV-derived DNA. This is a method for quantitatively evaluating the Rev nuclear export inhibitory activity of a test substance based on the measurement result.

  “HIV-derived DNA” is DNA prepared based on the known sequence of the HIV gene. A method in which HIV-derived DNA is inserted into a vector such as a plasmid and the vector is transfected into a host cell is preferred. Moreover, if the full length of viral DNA is used, HIV virus itself will be produced after transfection. Therefore, in order to avoid virus production, it is preferable to use, as the HIV-derived DNA, DNA obtained by removing all or part of any gene region from structural genes such as pol and env.

  In the present invention, the conditions required for the above-mentioned HIV-derived DNA (or a vector into which the DNA is inserted) include (1) Rev protein expression in the host cell upon introduction, and (2) RRE to which Rev binds ( (Rev response element) region is transcribed, and (3) a predetermined constituent protein (for example, p24) to be detected is expressed by Rev translocation into the nucleus.

  Various types, subtypes, and virus strains are known as HIV viruses, but the above HIV-derived DNA may be prepared based on any sequence. The type of vector to be used is not particularly limited as long as the above conditions are satisfied.

  The “transfection” method is not particularly limited, and a conventionally known transfection method (transformation method) such as an electroporation method, a calcium phosphate method, a liposome method, or a DEAE dextran method can be used. Among them, the calcium phosphate method is a transfection method used in Examples described later, but it is a simple and efficient method.

  In the examples described later, HeLa cells were used as “cells” (host cells). Of course, cell types other than HeLa cells may be used. When adopting the calcium phosphate method as the transfection method, it is preferable to use adherent cells, and examples thereof include the use of cell types such as KB, HepG2, MCF-7, T24, HT1080, and PC3.

  The “predetermined HIV constituent protein” to be detected may be a constituent protein translated from HIV-derived RNA transported to the outside of the nucleus by binding to Rev protein, but is a constituent unit of p55 which is a Gag protein. Yes, p24 having the same epitope can be mentioned as a suitable one. Regarding p24, there is also a commercially available detection kit using the ELISA method, which can be detected with high accuracy.

  In this assay method, the “expression level” of a constituent protein is measured on a cell sample. The cell sample is a cell lysate or a protein extract (crude protein solution) obtained by fractionating a cell lysate by centrifugation. ) Is preferably used.

  An ELISA method (enzyme linked immunosorbent assay) can be preferably used for measuring the “expression level”. In the ELISA method, the expression level of the target protein is measured and evaluated using the enzyme activity as an index. For example, when measuring the expression level of p24, first, an antigen-antibody reaction is performed using an anti-p24 antibody that specifically recognizes the antigen p24, and the antigen in the sample is bound to the antibody. Next, the enzyme reaction is allowed to proceed by adding the substrate to an enzyme (for example, horseradish peroxidase) directly or indirectly bound to the anti-p24 antibody. By measuring the absorbance of the sample using the color developed by the enzyme reaction, the expression level of p24 in the sample can be measured and evaluated.

  In the ELISA method, various modifications can be made with respect to the type of enzyme used, the method of binding the antibody and the enzyme, and any method may be used in the present invention. In addition to the ELISA method, an antibody against an HIV constituent protein to be detected is directly or indirectly labeled with a fluorescent substance or a chemiluminescent substance, and the protein is determined based on the fluorescence or chemiluminescence obtained from the sample. May be measured.

  When the expression level of the HIV constituent protein is small, it can be evaluated that the Rev nuclear export inhibitory activity of the test substance is high. On the other hand, when the expression level is large, the Rev nuclear export inhibition activity of the test substance is low. it can. This is due to the following reason.

  In general, large proteins that cannot pass through the nuclear pores by passive diffusion are transferred into or out of the nucleus by a transport carrier. The transported protein has a nuclear translocation signal (NLS) or a nuclear translocation signal (NES), and the transport carrier recognizes the signal and transports the protein into and out of the nucleus. Rev has a region that recognizes NES and HIV-derived RNA, and forms a complex called HIV-derived RNA-Rev-transport carrier and is transported out of the nucleus. Here, the transport carrier responsible for nuclear transfer is called CRM1.

  The HIV-derived RNA is usually translated after being spliced in the nucleus, but by binding Rev, it is transported out of the nucleus while avoiding splicing, and the constituent proteins are synthesized. That is, it can be said that Rev's nuclear translocation is not inhibited as the amount of constituent protein synthesized increases, while Rev's nuclear translocation is inhibited as the amount of constituent protein synthesized is small.

  As a method for quantitatively evaluating the Rev nuclear export inhibitory activity of a test substance, it is preferable to use the following method using a cell group having an inhibition rate of 0% and a cell group having an inhibition rate of 100% as controls. That is, the cells are divided into three groups, and the first group of cells is transfected with the HIV-derived DNA (that satisfies the above conditions (1) to (3)). The second group of cells is transfected with a different HIV-derived DNA. More specifically, the Rev protein is not expressed in the host cell without the above-mentioned condition (1), or the Rev protein that has lost its original function (function of transferring HIV-derived RNA to the nucleus) is expressed. HIV-derived DNA is transfected into the second group of cells.

  The third group of cells is administered the test substance after the same HIV-derived DNA as that of the first group of cells (that satisfies the conditions (1) to (3) described above) is transfected. After a predetermined period of time, the cells of each group are disrupted to prepare a protein extract, and the expression level of a predetermined HIV constituent protein (for example, p24) in each extract is measured by an ELISA method. Based on this, the Rev nuclear export inhibitory activity of the test substance is quantitatively evaluated. At this time, if the inhibition rate of the cells of the first group is 0% and the inhibition rate of the cells of the second group is 100%, the Rev nuclear export inhibition activity of the test substance (in other words, inhibition) Rate) can be quantitatively evaluated.

  In the above assay method, preferably, a foreign gene is introduced into each group of cells in addition to HIV-derived DNA, and a protein extract from each group of cells is diluted according to the expression level of the foreign gene, The expression level of a predetermined HIV constituent protein in these diluted solutions is measured. Thereby, the transfection efficiency can be normalized. In addition, it can be confirmed that general transcription and translation systems unrelated to this assay system are not inhibited.

  The foreign gene to be introduced is exemplified by a luciferase gene, but is not particularly limited. When the luciferase gene is introduced, the luciferase activity in each sample is measured according to a conventional method, and each sample may be diluted based on the measurement result.

  In addition, for introduction of the luciferase gene into the cell, a plasmid in which the gene is inserted may be used, but the sequence of the plasmid is preferably a sequence that has been confirmed to be independent of Rev.

  In the above assay method, HIV-derived DNA and a foreign gene may be introduced into a cell using a plasmid, and a plasmid for adjusting the amount of transfection may be further introduced into each group of cells. In the examples described later, PUC12 was introduced into each group of cells as a plasmid for adjusting the amount of transfection. However, the type of plasmid to be used is not particularly limited as long as this assay system is not affected. .

[2] Specific examples of the assay method of the present invention (Examples)
As a specific example (example) of the assay method of the present invention described above, the following assay system was assembled.
The HIV gene (HIV-derived DNA) is transfected into cells, and the constituent protein p24 expressed as a result is quantified by ELISA, thereby evaluating the Rev nuclear export inhibitory activity of the test substance. At the same time, in order to normalize the transfection efficiency and to confirm that it does not interfere with general transcription and translation systems in addition to this assay system, a luciferase gene-encoding plasmid is simultaneously transfected. . By such an assay system, a compound that does not suppress luciferase activity but suppresses p24 production as a Rev nuclear export inhibitor is searched.
Specifically, the Rev nuclear export inhibitory substance was assayed according to the following procedures 1 to 7.

[Method of assay]
Procedure 1: Cultured HeLa cells are seeded in a 6-well plate at 4 × 10 ⁴ cells / 3 mL / well, cultured at 37 ° C. and 5% CO ₂ , and the medium is replaced with fresh one after 21 hours. It was.

Procedure 2: 24 hours after seeding, the plasmid suspension is transfected by the calcium phosphate method. The following four types of plasmids were used.
(1) pcRRE: a plasmid into which an HIV-derived DNA from which the pol region has been deleted (or inactivated) from the HIV gene is introduced (2) pcRRE / Δrev: the rev gene region encoding the Rev protein from the plasmid pcRRE is missing (or not) Activated plasmid (3) luc: a plasmid encoding a luciferase gene and incorporating an RSV promoter upstream thereof (4) PUC12: a plasmid used to regulate the total amount of DNA to be transfected. It is a general cloning vector.
Then, the cells were divided into Group A and Group B, and the cells of each group were transfected by appropriately combining the plasmids (1) to (4). Specifically, pcRRE, luc and PUC12 were transfected into group A cells, and pcRRE / Δrev, luc and PUC12 were transfected as controls for group B cells.

Procedure 3: After culturing at 37 ° C. and 5% CO ₂ for 16 hours after transfection, the cells were washed twice with a medium to remove calcium phosphate precipitates.
Next, replace with fresh medium, divide group A into test substance administration group and non-administration group, and add DMSO solution of test substance to the final concentration of DMSO to the cells of the administration group did. On the other hand, only DMSO was added to the non-administered group of Group A and the cells of Group B.

Procedure 4: After addition of the test substance, the cells were cultured at 37 ° C. and 5% CO ₂ for 12 hours. Thereafter, the medium was removed, washed with PBS, and lysis bufer was added to disrupt each group of cells. The cell lysate was collected, the insoluble fraction was precipitated by centrifugation, and the supernatant was used as a crude protein solution.

  Procedure 5: The luciferase activity of the crude protein solution of each group was measured according to a conventional method.

Procedure 6: After diluting the crude protein solution based on the luciferase activity, the production amount of p24 which is an HIV constituent protein was measured in each group. The p24 was measured using a commercially available p24 ELISA kit (manufactured by ZeptoMetrix) according to the method described in the kit manual.
The crude protein solution of each group was diluted as follows so that the luciferase activity became a constant value for each group. For example, when diluting to luciferase activity 10,000 RLU / 10 μL, if group A activity: 25,000 RLU / 10 μL, group B activity: 500,000 RLU / 10 μL, group C activity: 125,000 RLU / 10 μL, then A Group: 2.5 times, B group: 50 times, C group: The expression level of p24 was evaluated using the crude protein solution diluted 12.5 times.

  Procedure 7: The amount of p24 produced in the group A supplemented with DMSO only (that is, the test substance non-administered group) is set to a value at which Rev nuclear translocation is not inhibited at all (0% inhibition), and the amount of p24 produced in group B is As the value (100% inhibition) when Rev was completely suppressed, the Rev nuclear export inhibitory activity of each test substance was evaluated from the value of p24 production in the test substance administration group.

[Result of assay]
The results of the assay performed by the assay system of this example are shown in Table 1.

  In the table, “luciferase” is a measurement result of luciferase activity (unit: RLU / 10 μL), “p24 production amount” is a result of measuring p24 production amount by the above ELISA method (unit pg / mL), “inhibition rate” These are the results of calculating and evaluating the inhibition rate of each test substance (Rev nuclear transfer inhibition rate) from this p24 production amount.

  “No treatment (pcRRE)” is the result of the non-test substance administration group among the group A transfected with the plasmid pcRRE, and the production amount of p24 at this time is set to a suppression rate of 0%. On the other hand, “No treatment (pcRRE / Δrev)” is the result of group B transfected with the plasmid pcRRE / Δrev, and the amount of p24 produced at this time is set to a suppression rate of 100%.

  “LMB” (leptomycin B) is a known Rev nuclear export inhibitor. As described above, a protein called CRM1 is known as a transport carrier responsible for nuclear export, and Rev is also bound to CRM1 and transported out of the nucleus. LMB has been shown to form a covalent bond with the Cys residue at position 529 of CRM1 and inactivate CRM1.

  Also in the assay system of this example, a significant decrease in the amount of p24 produced was observed when LMB was allowed to act. The inhibition rate at this time was evaluated as 92.1%. That is, the inhibition of Rev translocation into the nucleus occurred, and the degree of inhibition was quantified and could be grasped as a numerical value.

  “Sample A · B” in the table is a test substance used in the assay of this example. Of both samples, sample B showed almost no inhibitory activity (suppression rate 6.7%), but sample A showed an inhibitory effect (suppression rate 41.2%).

  As described above, according to the assay system of this example, the Rev nuclear export inhibitory activity of each test substance could be quantified, and good results were obtained in terms of reproducibility.

  In the assay method of the present example, each culture period of the cells may be appropriately changed, but the culture period from the transfection of the plasmid into the cells until the sample treatment (test substance administration) is about 16 hours. It is desirable to set to. This is because the time when the transfected DNA is in the cell but the expression of the Gag protein p24 has not started is about 16 hours.

  The culture for 12 hours after the addition of the test substance is set to a time when it is difficult for the cytotoxicity to occur and the suppression of Gag production is easy to see. Moreover, regarding pre-culture before transfection, conditions under which transfection efficiency was good are employed, but the conditions may be changed as appropriate.

[3] Assay Kit, Assay Device The assay method of the present invention can be made into a kit or used as an assay device. In the case of making a kit, in addition to the plasmids used in the above examples, various reagents, enzymes, buffers, antibodies, medium components, etc. can be included in the kit, and further combined with instruments such as plates to make an assay device Can do.

As described above, the present invention relates to a novel assay method for a Rev protein nuclear translocation regulator, and is preferably used to search for an anti-HIV drug whose action mechanism is inhibition of Rev protein nuclear translocation. Can be used for research purposes for other purposes.

Claims (12)

  After transfection with DNA derived from HIV, the expression level of a predetermined HIV-constituting protein is measured for the cells to which the test substance is administered, and the Rev nuclear export inhibitory activity of the test substance is quantitatively determined based on the measurement result. A method for assaying a Rev nuclear export control substance, characterized by comprising:   The assay method according to claim 1, wherein the expression level of p24 is measured as a predetermined HIV constituent protein.   The assay method according to claim 2, wherein the expression level of p24 is measured by an ELISA method. A first group of cells in which the RRE region is transcribed into RNA in a host cell and transfected with HIV-derived DNA that expresses the Rev protein;
A second group of cells transfected with HIV-derived DNA that does not express Rev protein in host cells or express Rev protein that has lost its original function; and
After the same HIV-derived DNA as that of the first group of cells was transfected, the expression level of a predetermined HIV constituent protein was measured for each of the third group of cells to which the test substance was administered. A method for assaying a Rev nuclear export control substance, characterized in that the Rev nuclear export inhibitory activity of a test substance is quantitatively evaluated.   The assay method according to claim 4, wherein the expression level of p24 is measured as a predetermined HIV constituent protein.   6. The assay method according to claim 5, wherein the expression level of p24 is measured by an ELISA method.   The first group of cells has an inhibition rate of 0%, the second group of cells has an inhibition rate of 100%, and quantitatively evaluates the Rev nuclear export inhibitory activity of the test substance by comparison with both. The assay method according to claim 4.   A foreign gene is introduced into each group of cells in addition to the HIV-derived DNA, and a protein extract from each group of cells is diluted according to the expression level of the foreign gene, and a predetermined HIV configuration in these dilutions The assay method according to claim 7, wherein the expression level of the protein is measured.   The assay method according to claim 8, wherein a luciferase gene is introduced as a foreign gene.   The assay method according to claim 8 or 9, wherein an HIV-derived DNA and a foreign gene are introduced into a cell by a plasmid, and a plasmid for adjusting the transfection amount is further introduced into each group of cells.   The kit for assay used for the assay method of any one of Claims 1-10. The apparatus for assay used for the assay method of any one of Claims 1-10.